Materials for peripheral nerve regeneration

Recent efforts in scientific research in the field of peripheral nerve regeneration have been directed towards the development of artificial nerve guides. We have studied various materials with the aim of obtaining a biocompatible and biodegradable two layer guide for nerve repair. The candidate materials for use as an external layer for the nerve guides were poly(caprolactone) (PCL), a biosynthetic blend between PCL and chitosan (CS) and a synthesised poly(ester-urethane) (PU). Blending PCL, which is a biocompatible synthetic polymer, with a natural polymer enhanced the system biocompatibility and biomimetics, fastened the degradation rates and reduced the production costs. Various novel block poly(ester-urethane)s are being synthesised by our group with tailored properties for specific tissue engineering applications. One of these poly(ester-urethane)s, based on a low molecular weight poly(caprolactone) as the macrodiol, cycloesandimethanol as the chain extender and hexamethylene diisocyanate as the chain linker, was investigated for the production of melt extruded nerve guides. We studied natural polymers such as gelatin (G), poly(L-lysine) (PL) and blends between chitosan and gelatin (CS/G) as internal coatings for nerve guides. In vitro and in vivo tests were performed on PCL guides internally coated either with G or PL to determine the differences in the quality of nerve regeneration associated with the type of adhesion protein. CS/G natural blends combined the good cell adhesion properties of the protein phase with the ability to promote nerve regeneration of the polysaccharide phase. Natural blends were crosslinked both by physical and chemical crosslinking methods. In vitro neuroblast adhesion tests were performed on CS/G film samples, PCL/CS and PU guides internally coated with G to evaluate the ability of such materials towards nerve repair.     

Tissue engineering and peripheral nerve regeneration (III)

The biodegradation rate and biocompatibility of poly (d, / -lactide) (PDLLA)in vivo were evaluated. The aim of this study was to establish a nerve guide constructed by the PDLLA with 3-D microenvironment and to repair a 10 mm of sciatic nerve gap in rats. The process of the nerve regeneration was investigated by histological assessment, electrophysiological examination, and determination of wet weight recovery rate of the gastrocnemius muscle. After 3 weeks, the nerve guide had changed from a transparent to an opaque status. The conduit was degraded and absorbed partly and had lost their strength with breakage at the 9th week of postoperation. At the conclusion of 12 weeks, proximal and distal end of nerves were anastomosed by nerve regeneration and the conduit vanished completely. The results suggest that PDLLA conduits may serve for peripheral nerve regeneration and PDLLA is a sort of hopeful candidate for tissue engineering.     

Preparation of chitosan/hydroxyapatite guided membrane used for periodontal tissue regeneration

<正>In an effort to develop biomaterials to meet guided tissue regeneration(GTR) standards for periodontal tissue recovery,a homogeneous and transparent chitosan(CS)/hydroxyapatite(HA) membrane with potential applications as GTR barrier in periodontal therapy has been prepared via in situ compositing.The membrane has been designed to have a smoothrough asymmetric structure that meets the demand for GTR.Component and morphology of the membrane are characterized by XRD and SEM.It can be indicated that HA was in situ synthesized uniformly in the CS membrane.Mechanical experiments of the membranes with various HA contents show that their tensile strengths are adequate for periodontal therapy.Biological properties of the membrane have been performed by cell toxicity assays,hemolysis tests and animal experiments.Results indicate that the membrane has good biocompatibility and inductive effect for cell growth.Therefore this membrane can be potentially applied as GTR barrier membrane for periodontal tissue regeneration.     

Preparation and characterization of microwave-treated carboxymethyl chitin and carboxymethyl chitosan films for potential use in wound care application

CM-chitin and CM-chitosan films were successfully crosslinked by microwave treatment. Crosslinking of the microwave-treated CM-chitin films involved mainly the carboxylate and the secondary alcohol groups, while crosslinking of microwave-treated CM-chitosan films involved the carboxylate and the amino groups. In addition, the crystallinity of CM-chitin increased with increasing microwave treatment time, whereas an increase in the crystallinity of the microwave-treated CM-chitosan films was not observed. At a similar percentage of weight loss, the crosslinking of either CM-chitin or CM-chitosan films by microwave treatment required much less stringent condition when compared with the crosslinking by autoclave treatment. Based on both direct and indirect cytotoxicity assays, the cytotoxicity of the microwave-treated CM-chitin films was negative, while that of the microwave-treated CM-chitosan films was positive. Human fibroblasts adhered on the surface of microwave-treated CM-chitosan films much better than on the surface of microwave-treated CM-chitin films.     

Preparation of enzymatically cross-linked sulfated chitosan hydrogel and its potential application in thick tissue engineering

For the requirement of preliminary vascularization, the scaffolds for thick tissue engineering should possess not only good cell affinity, but also anticoagulant ability. In this paper, an enzymatically crosslinked hydrogel scaffold based on sulfated chitosan (SCTS) was prepared. Firstly, sulfated chitosan-hydroxyphenylpionic acid (SCTS-HPA) conjugate was synthesized, and its structure was identified by FITR and ¹H NMR. And then an enzymatically crosslinked hydrogel was prepared in the presence of horseradish peroxidase (HRP) and hydrogen peroxide (H₂O₂). The gelation time, mechanical property, morphology and cytotoxicity to human umbilical vein endothelial cells (HUVECs) of the hydrogel was evaluated in vitro, the tissue compatibility of SCTS scaffold was studied in vivo. The results showed that the gelation time, mechanical property, morphology of the dehydrated hydrogel could be controlled by the HRP and H₂O₂ concentration. The cytotoxicity test showed that the hydrogel extracts had no cytotoxicity to HUVECs. The in vivo assay indicated that SCTS-HPA scaffold showed good tissue compatibility, and no thrombus formation. All these results indicated that the SCTS-HPA scaffold could be used as thick tissue engineering scaffold.     

Synthesis and characterization of thermoresponsive hydrogels cross-linked with chitosan

Hydrogels composed of N-isopropylacrylamide (NIPAAm) and acrylic acid (AAc) were prepared by redox polymerization with degradable chitosan cross-linkers. Chitosan degradable cross-linkers were synthesized by the acrylation of the amine groups of glucosamine units within chitosan and characterized with ¹H NMR. With the chitosan cross-linkers, loosely cross-linked poly(N-isopropylacryamideco-acrylic acid) [P(NIPAAm-co-AAc)] hydrogels were prepared, and their phase transition behavior, lower critical solution temperature (LCST), water content and degradation properties were investigated. The chitosan cross-linked P(NIPAAm-co-AAc) hydrogels were pliable and transparent at room temperature. The LCST could be adjusted at 32∼39°C by alternating the feed ratio. Swelling was influenced by NIPAAm/AAc monomer ratio, cross-linking density, swelling media, and temperature. All hydrogels with different feeding ratios contained more than 95% water at 25°C in the ultra pure water and phosphate-buffered saline (PBS, pH = 7.4 ± 0.1), and had a prospective swelling in the simulated gastric fluids (SGF, pH = 1.2) > 72.54%. In degradation studies, breakdown of the chitosan cross-linked P(NIPAAm-co-AAc) hydrogels was dependent on the cross-linking density. The chitosan cross-linked P(NIPAAm-co-AAc) hydrogels which can be tailored to create environmentally-responsive artificial extracellular materials have great potential for future use.      

Self-aggregated nanoparticles from linoleic acid modified carboxymethyl chitosan: Synthesis, characterization and application in vitro

The purpose of the present research work was to study the formation of linoleic acid (LA) modified carboxymethyl chitosan (LCC). Another objective was to evaluate effect of linoleic acid degree of substitution on loading capacity (LC), ADR loading efficiency (LE) and in vitro release profile of LCC nanoparticles. The hydrogel nanoparticles can be prepared using linoleic acid modified carboxymethyl chitosan (LACMCS) after the sonication. The critical aggregation concentration (CAC) of the self-aggregate of LA modified CMCS (LCC) was determined by measuring the fluorescence intensity of the pyrene as a fluorescent probe. The CAC values were in the range of 0.061–0.081 mg/mL. Self-aggregated nanoparticles exhibited an increased LC and LE, decreased sustained release with an increasing ratio of the hydrophobic LA to hydrophilic CMCS. LCC nanoparticles loaded with ADR exerted in vitro anticancer activity against Hela cells that was comparable to the activity of free (non-entrapped in nanoparticles) ADR.     

Cell outer membrane mimetic modification of a cross-linked chitosan surface to improve its hemocompatibility

A novel strategy has been developed to improve the hemocompatibility of chitosan surface by cell outer membrane mimetic structure able to reduce protein adsorption and cell adhesion. Phosphorylcholine dichloride was synthesized and grafted onto a glutaraldehyde-cross-linked chitosan (CS-GA) film surface to prepare phosphorylcholine-coated CS-GA film (CS-GA-PC) through a heterogeneous reaction process. The spectroscopic and contact angle characterization show that a cell outer membrane mimetic structure was formed on the cross-linked chitosan surface, and the significantly improved hemocompatibility of the modified surface was shown by a suppression of 94% on platelet adhesion, a suppression of 60–70% for bovine plasma fibrinogen and bovine serum albumin adsorptions. These results demonstrated that this cell outer membrane mimetic surface modification with phosphorylcholine dichloride is a promising strategy to improve the hemocompatibility of chitosan.     

In vitro biocompatibility of electrospun and solvent-cast chitosan substrata towards Schwann, osteoblast, keratinocyte and fibroblast cells

Chitosan or poly(N-acetyl-d-glucosamine-co-d-glucosamine) with a degree of deacetylation of about 85% was fabricated into nanofibrous membranes by electrospinning from 7% w/v chitosan solution in 70:30 v/v trifluoroacetic acid/dichloromethane solvent system. The obtained fibers were smooth without the presence of beads. The diameters of the individual fiber segments were 126 ± 20 nm. The potential for use of the electrospun chitosan nanofibrous membranes as substrates for cell/tissue culture was evaluated with four different cell types, i.e., Schwann cells, osteoblast-like cells, keratinocytes and fibroblasts, in terms of the attachment and the proliferation of the cells as well as the morphology of the seeded and the cultured cells. The results were compared with the corresponding solvent-cast films. Both types of the chitosan substrates supported the attachment and, at the same time, promoted the largest increase in the viability of the cultured keratinocytes. The viability of Schwann cells cultured on these substrates increased marginally well, but the attachment of the cells on the surfaces was relatively poor. Finally, both types of the chitosan substrates showed cytostatic property towards both osteoblast-like cells and fibroblasts, despite the convincingly good attachment of osteoblast-like cells on the surfaces.     

pH and ionic sensitive chitosan/carboxymethyl chitosan IPN complex films for the controlled release of coenzyme A

pH and ionic sensitive interpenetrating polymer network (IPN) complex films based on chitosan (CS) and carboxymethyl chitosan (CM-CS) were prepared by using glutaraldehyde as crosslinking agent. Its structure was characterized by FT-IR, which indicated that the IPN was formed. The films were studied by swelling, weight loss with time, and release of coenzyme A (CoA). It was found that the IPN films were sensitive to pH and ionic strength of the medium. The cumulative release rate of CoA decreased with CoA loading content, ionic strength or crosslinking agent increasing. The composition of the IPN films and pH of release medium also had significant effect on the release of CoA. The differences in the rates and amounts of released CoA may be attributed to the swelling behavior, the degradation of films, and interaction between drug molecule and polymer matrix. These results suggested CS/CM-CS IPN films could be used as drug delivery carrier.     

交联羧甲基魔芋葡甘聚糖空心微球制备及应用

以魔芋葡甘聚糖(KGM)为壁材,通过羧甲基化并经乳状液化学交联制备球体较为完好的羧甲基魔芋葡甘聚糖(CMKGM)空心微球.通过扫描电镜(SEM)和透射电镜(TEM)对CMKGM空心微球的形貌和粒径进行了表征.结果表明,KGM溶胶浓度为0.5%-1%、乳化剪切速率为8000r/min、环氧氯丙烷(ECH)为5ml时,可以得到粒径均匀、形貌规整、溶胀性能良好的CMKGM空心微球.增大溶液的pH和羧甲基试剂的用量均有利于CMKGM空心微球对水中Cu2+的吸附.     

New approach to the quaternization of chitosan and its amphiphilic derivatives

Regioselective quaternization of chitosan and its amphiphilic derivatives has been carried out by means of reaction with betaine in the presence of the coupling reagent 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) in aqueous media at pH 5.5 ± 0.5. This reaction results in preparation of N-/(trimethylammonio)acetyl/chitosan chloride and its amphiphilic derivatives. The degree of quaternization increases with increasing EEDQ/chitosan ratio and is partly accompanied by N-ethoxycarbonylation. That side-product formation can be minimized by increasing betaine/EEDQ ratio.     

Glutaraldehyde cross-linked epoxyaminated chitosan as an adsorbent for the removal and recovery of copper(II) from aqueous media

A novel glutaraldehyde cross-linked epoxyaminated chitosan (GA-C-ENCS) prepared through chemical modification was used as an adsorbent for the removal and recovery of Cu(II) from aqueous media. The adsorbent was characterized by FTIR, SEM-EDS, ESR, TG/DTG, BET-surface area and potentiometric titration. The Cu(II) adsorption process, which was pH dependent showed maximum removal at pH 6.0. Adsorption equilibrium was achieved within 3 h. The adsorption of Cu(II) followed a reversible-first-order kinetics. The equilibrium data were evaluated using the Langmuir, Freundlich and Dubinin–Radushkevich isotherm models. The best interpretation for the equilibrium data was given by the Dubinin–Radushkevich isotherm. The adsorption capacity of the adsorbent increased from 3.11 to 3.71 mmol g⁻¹ when the temperature was increased from 20 to 50 °C. The complete removal of 20.7 mg L⁻¹ Cu(II) from electroplating industry wastewater was achieved by 0.4 g L⁻¹ GA-C-ENCS. Regeneration experiments were tried for four cycles and the results indicate a capacity loss of <7.0%.     

Shell cross-linked stearic acid grafted chitosan oligosaccharide self-aggregated micelles for controlled release of paclitaxel

Stearic acid grafted chitosan oligosaccharide (CSO-SA) with different degree of amino substitution (SD) was synthesized by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-mediated coupling reaction. The critical micelle concentration (CMC) of CSO-SA with different SD was about 0.06, 0.04, 0.01 mg/ml, respectively. With the increase of micelle concentration, the micelle size decreased, and the zeta potential increased. On the other hand, with the increase of SD of CSO-SA, the micelle size and zeta potential decreased due to the increased hydrophobic interaction of SA and the reduced free amino groups. To increase the stability of the micelle in vivo and controll drug release, the shells of micelles were cross-linked by glutaraldehyde. By controlling the molar ratio of CSO-SA to glutaraldehyde, the cross-linking of intra-micelle could be reached, and the nanoparticle with smaller size than that of its initial micelle was obtained. Paclitaxel was then used as model drug to incorporate into the micelles, and the surfaces of the micelles were further cross-linked by glutaraldehyde to form drug loaded and shell cross-linked nanoparticles. The effects of drug loading, SD of CSO-SA and cross-link degree on the size, zeta potential, drug entrapment efficiency and in vitro drug release behavior of micelles and its cross-linked nanoparticles were investigated. The higher drug entrapment efficiencies (above 94%) were observed in all case. The charged amounts of drug did not affect the drug release behavior. The drug release rate decreased with the increase of SD of CSO-SA and cross-link degree.     

Determination of the viscometric constants for chitosan and the application of universal calibration procedure in its gel permeation chromatography

The viscometric constantsa andK in the Mark-Houwink equation were determined in 0.5 M acetic acid-0.5 M.sodium acetate solution for chitosan fractionated by gel filtration. The weight-average molecular weight of each fraction was measured by the light-scattering method. The values obtained area=0.59 andK=0.119 cm³ g^–1.The molecular weightsMw andMn for fractionated chitosan were measured by GPC. The value ofMw by GPC was much different from that by light scattering and, therefore, a universal calibration procedure was applied to the data by GPC. It was concluded that, also in the case of a cationic polysaccharide such as chitosan, the universal calibration procedure is effective for obtaining the reliable molecular weight by GPC.     

In vivo evaluation of a biodegradable EDC/NHS-cross-linked gelatin peripheral nerve guide conduit material

Peripheral nerve regeneration has been evaluated using a biodegradable nerve conduit, which is made of a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS) cross-linked gelatin. The EDC/NHS crosslinked gelatin (ENG) conduit is brownish in appearance, and is concentric and round with a smooth outer surface and inner lumen. After subcutaneous implantation on the dorsal side of a rat, the degraded ENG conduit only evoked a mild tissue response, with the formation of a thin tissue capsule surrounding the conduit. Biodegradability of the ENG conduit and its effectiveness as a guidance channel has been examined by its use to repair a 10 mm gap in the rat sciatic nerve. As a result, the tubes degraded throughout the implantation period, but still remained circular with a thin round lumen until they were completely integrated with the enclosed nerves. Successful regeneration through the gap occurred in all the conduits over the three experimental periods of 4, 8, and 12 weeks. Histological observation showed that numerous myelinated axons had crossed through the gap region even at the shortest implantation period of 4 weeks. Peak amplitude, area under the muscle action potential curve, and nerve conductive velocity all showed an increase as a function of the recovery period, which indicates that the nerve had undergone adequate regeneration. These results indicate the superiority of the ENG materials and suggest that the novel ENG conduits provide a promising tool for neuro-regeneration.     

New potential anti-SARS-CoV-2 and anti-cancer therapies of chitosan derivatives and its nanoparticles: Preparation and characterization

Chitosan (CS) is a biopolymer and has reactive amine/hydroxyl groups facilitated its modifications. The purpose of this study is improvement of (CS) physicochemical properties and its capabilities as antiviral and antitumor through modification with 1-(2-oxoindolin-3-ylidene)thiosemicarbazide (3A) or 1-(5-fluoro-2-oxoindolin-3-ylidene)thiosemicarbazide (3B) via crosslinking of poly(ethylene glycol)diglycidylether (PEGDGE) using microwave-assisted as green technique gives (CS-I) and (CS-II) derivatives. However, (CS) derivatives nanoparticles (CS-I NPs) and (CS-II NPs) are synthesized via ionic gelation technique using sodium tripolyphosphate (TPP). Structures of new (CS) derivatives are characterized using different tools. The anticancer, antiviral efficiencies and molecular docking of (CS) and its derivatives are assayed. (CS) derivatives and its nanoparticles show enhancement in cell inhibition toward (HepG-2 and MCF-7) cancer cells in comparison with (CS). (CS-II NPs) reveals the lowest IC₅₀ values are 92.70 ± 2.64 μg/mL and 12.64 µ g/mL against (HepG-2) cell and SARS-CoV-2 (COVID-19) respectively and the best binding affinity toward corona virus protease receptor (PDB ID 6LU7) ?5.71 kcal / mol. Furthermore, (CS-I NPs) shows the lowest cell viability% 14.31 ± 1.48 % and the best binding affinity ?9.98 kcal/moL against (MCF-7) cell and receptor (PDB ID 1Z11) respectively. Results of this study demonstrated that (CS) derivatives and its nanoparticles could be potentially employed for biomedical applications.     

羧甲基壳聚糖制备中的溶剂影响及精制方法研究

研究了有机溶剂中两步法制取羧甲基壳聚糖的反应过程.采用有机溶剂溶胀、氢氧化钠碱化、氯乙酸改性、乙醇溶析等步骤制备了高取代度羧甲基壳聚糖.考察了碱用量、氯乙酸用量和有机溶剂类型三个因素对羧甲基壳聚糖取代度的影响.通过比较二甲基亚砜、异丙醇、丙酮和乙醇四种有机溶剂中壳聚糖羧甲基化取代度变化规律,分析了羧甲基化的机理.实验结...     

Adsorption of Cu2+ by modified chitosan microspheres and its application in homocoupling of arylboronic acid

The CC bond coupling reactions have a wide range of applications in the synthesis of natural substances and the synthesis of physiologically active compounds. We first prepared modified chitosan microspheres by emulsification and investigated the performance of the chitosan adsorbent for the adsorption of copper ions. We then evaluated the catalytic performance of chitosan-supported copper microspheres in the self-coupling of arylboronic acids using 4-methoxybenzene as the model substrate, while exploring a wide range of substrates including aryl boronic acid derivatives and heterocyclic arylboronic acids, including aldehyde, nitro, and aldehyde groups. The experimental results show that good to excellent yields (55%-95%) of the desired dimer products were obtained under mild conditions at room temperature and in air, and the catalytic material could be reused up to five times with no significant decrease in catalytic activity.     

Oxalic acid cross-linked sodium alginate and carboxymethyl chitosan hydrogel membrane for separation of dye/NaCl at high NaCl concentration

Dye desalination is a challenge in the treatment of textile wastewater with high salt concentration. It is imperative to develop salt resistance membrane that is from sustainable materials to effectively treat dye/salt mixtures. And most polymer membrane materials are non-renewable petrochemical resources.In this paper, a green hydrogel membrane(CMCS-OA-Na Alg) was prepared by non-metallic ions of oxalic acid(OA) cross-linking of two natural macromolecules of sodium alginate(Na Alg) and carboxym...